DNA purification by solid phase extraction using fluorinated celite

ABSTRACT

The present invention relates to fluorinated surfaces which exhibit sufficient hydrophilicity and sufficient electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the surface. Generally, the hydrophilic and electropositive characteristics are expressed at the fluorinated surface. Preferred fluorinated surfaces of the present invention include fluorinated Al(OH) 3 , fluorinated SiO 2  and fluorinated Celite. The fluorinated surfaces of the present invention are particularly useful in processes for purification of DNA from other cellular components. In these processes, a suspension of cellular components is placed in contact with the fluorinated surface, the fluorinated surface is washed to remove all cellular components other than DNA which are bound to the surface, and the bound DNA is eluted from the surface. Lower concentrations of chaotrope in the binding buffer are needed to bind DNA to the fluorinated surfaces.

This is a division of application Ser. No. 08/127,407, filed Sep. 27,1993, now U.S. Pat. No. 5,438,129.

BACKGROUND OF THE INVENTION

The present invention relates generally to the purification of DNA bysolid phase extraction, and more specifically to fluorinated surfaceswhich are capable of binding DNA and eluting DNA under suitableconditions.

The preparation of high-purity double-stranded (ds) plasmid DNA,single-stranded (ss) phage DNA, chromosomal DNA and agarose gel-purifiedDNA fragments is of critical importance in molecular biology. Ideally, amethod for purifying DNA should be simple, rapid and require little, ifany, additional sample manipulation. DNA rendered by such a methodshould be immediately amenable to transformation, restriction analysis,ligation or sequencing. A method with all of these features would beextremely attractive in the automation of DNA sample preparation, a goalof research and diagnostic laboratories. Typically, the preparation ofplasmid DNA from crude alcohol precipitates is laborious, most oftenutilizing CsCl gradients, gel filtration, ion exchange chromatography,or RNase, proteinase K and repeated alcohol precipitation steps. Thesemethods also require considerable downstream sample preparation toremove CsCl and other salts, ethidium bromide and alcohol. Similararguments extend when using any of these methods for purifying DNAfragments. A further problem with these methods is that small,negatively-charged cellular components can co-purify with the DNA. Thus,the DNA can have an undesirable level of contamination.

DNA can also be purified using solid phases. Conventional solid phaseextraction techniques have utilized surfaces which either (1) fail toattract and hold sufficient quantities of DNA molecules because ofsurface design to permit easy recovery of the DNA molecules duringelution, or (2) excessively adhere DNA molecules to the surface, therebyhindering recovery of the DNA molecules during elution. Conventionalmetal surfaces which cause these problems when utilized in solid phaseextraction include silica surfaces such as glass and Celite. Adequatebinding of DNA to these types of surfaces can be achieved only byutilizing high concentrations of chaotropes or alcohols which aregenerally toxic, caustic, and/or expensive. For example, it is knownthat DNA will bind to crushed glass powders and to glass fiber filtersin the presence of chaotropes. The chaotropic ions typically are washedaway with alcohol, and the DNAs are eluted with low-salt solutions orwater. Importantly, RNA and protein do not bind. However, a seriousdrawback in the use of crushed glass powder is that its binding capacityis low. In addition, glass powders often suffer from inconsistentrecovery, incompatibility with borate buffers and a tendency to nicklarge DNAs. Similarly, glass fiber filters provide a nonporous surfacewith low DNA binding capacity. Other silicas, such as silica gel andglass beads, are not suitable for DNA binding and recovery. Currently,the solid phase of choice for solid phase extraction of DNA is Celitesuch as found in Prep-A-Gene™ by Bio-Rad Laboratories. As with thecrushed glass powders, high concentrations of chaotropes are requiredfor adequate binding of the DNA to the Celite.

SUMMARY OF THE INVENTION

These problems with conventional DNA purification methods are addressedby the present invention, which relates to fluorinated surfaces whichexhibit sufficient hydrophilicity and sufficient electropositivity tobind DNA from a suspension containing DNA and permit elution of the DNAfrom the surface. Generally, the hydrophilic and electropositivecharacteristics are expressed at the fluorinated surface, and arequantified as the presence of oxygen as measured by Fourier transforminfrared spectroscopy (FTIR) and the presence of the substituted atom asdetected by electron surface composition analysis (ESCA). Preferredfluorinated surfaces of the present invention include fluorinatedAl(OH)₃, fluorinated SiO₂ and fluorinated Celite.

The fluorinated surfaces of the present invention are particularlyuseful in processes for purification of DNA from other cellularcomponents. In these processes, a suspension of cellular components isplaced in contact with the fluorinated surface, the fluorinated surfaceis washed to remove all cellular components other than DNA which arebound to the surface, and the bound DNA is eluted from the surface.Lower concentrations of chaotrope in the DNA binding buffer are neededto bind DNA to the fluorinated surfaces.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to fluorinated surfaces which exhibitsufficient hydrophilicity and sufficient electropositivity to bind DNAfrom a suspension of cellular components and permit elution of the DNAfrom the surface. It has been found that much lower concentrations ofchaotropes or alcohols can be utilized to achieve purification of DNAusing the instant fluorinated surfaces.

DNA interacts with a solid phase surface in two ways. First, DNAinteracts with the surface through hydrogen bonding between phosphategroups of DNA and surface components of the solid phase, such as surfacehydroxyls. The second interaction is between the negatively chargedphosphates of the DNA and positively charged elements of the solid phasesurface. The hydrophilic and electropositive characteristics of thesolid phase surface must be such as to allow binding of the DNA from asuspension of cellular components, a suspension of nucleic acids andother materials, and/or a suspension of nucleic acids, and to permitelution of the DNA from the solid phase surface. Thus, theelectro-positive characteristics of the solid phase surface cannot havetoo high of a positive charge, or the DNA will stick to the surface andcannot be eluted. This characteristic is also true for many metal-basedsurfaces, which has resulted in their inability to be utilized forpurification of DNA.

Silicon-containing materials, e.g., silica, Celite, glass powders andthe like, have been used for DNA purification with mixed results. Someof these surfaces have low binding capacities and/or require the use ofhighly concentrated solutions of chaotropes or alcohols for the bindingof DNA. Other surfaces, such as Al(OH)s, have been found to bind almostone hundred percent of DNA in a suspension, but not to elute the boundDNA. Thus, it is desired to produce solid phase surfaces, particularlysolid phases of fluorinated surfaces, which exhibit suitable hydrophilicand electropositive characteristics for DNA purification and/or for DNApurification with much lower concentrations of chaotropes or alcohols.On the surface of the solid phase, hydrophilic characteristics areachieved by the presence of groups that will attract water molecules.Suitable groups include --OH, --NH, --F, --H or groups withdouble-bonded oxygen such as carbonyl, sulfonyl or phosphonyl.Electropositive characteristics are achieved by the presence ofpositively charged atoms. Suitable positively-charged atoms include Si,B or Al. In accordance with the present invention, fluorinated surfacesare prepared in which the hydrophilic characteristics are achieved byincorporation of fluorine groups, and the electropositivecharacteristics are achieved by incorporation of Si, Al or otherappropriate positively-charged atoms. Preferred fluorinated surfaces ofthe present invention include fluorinated Al(OH)₃, fluorinated SiO₂ andfluorinated Celite.

In general, the fluorinated surfaces of the present invention areprepared by reacting a suitable fluoride with the desired surface. Anyfluoride, preferably sodium fluoride and tetrabutylammonium fluoride,may be utilized in this reaction. It is preferred to usetetrabutylammonium fluoride. Suitable surfaces include those which bindDNA but fail to elute it. Such surfaces include Al(OH)₃, SiO₂, Celite,or any other solid that contains electropositive elements which aresubject to nucleophilic attack by fluoride. Surfaces with differentamounts of fluoride on the surface are prepared by reacting differentproportions of fluoride and the surface. In general, the fluoride isadded to the solid surface. A suitable solvent such as tetrahydrofuran(THF) is added and the reaction mixture is preferably refluxedovernight, although the reaction can be refluxed longer than 24 hours.More THF is added and the mixture heated at the above temperatureovernight to keep wet. Alternatively, the reaction mixture could simplybe heated overnight. The fluorinated surface is filtered, washed,air-dried briefly and oven-dried at 100° C. for ˜1 hour. The fluorinatedsurface is then stored in a desiccator.

Fluorinated Al(OH)₃ surfaces are prepared as generally described above,preferably by refluxing. DNA normally binds tightly to the untreatedAl(OH)₃ surface and is retained during elution. The presence of fluorinecauses less tight bonding of DNA to the treated Al(OH)₃ surface due torepulsion of F(δ-) and the phosphate back bone of DNA, so that the boundDNA would elute from the fluorinated Al(OH)₃ surface during the elutionstep. In general, as the percentage of fluorine on the Ai(OH)sincreases, the elution of DNA from the treated surface also increases.Fluorinated Al(OH)₃ surfaces prepared by reacting about 0.05 to about1.5 equivalent of fluorine to Al(OH)₃ were found to provide goodrecovery of DNA from biological samples. It is preferred that thefluorinated Al(OH)₃ surfaces be prepared by reacting about 0.3 to about0.9 equivalent of fluorine to Al(OH)₃, and most preferred that they beprepared by reacting about 0.3 equivalent of fluorine to Al(OH)₃. ForDNA recovery, Al(OH)₃ fluorinated with 0.3 equivalent of fluorideout-performs super fine super floss Celite.

Fluorinated rehydrated Celite surfaces are prepared as generallydescribed above, preferably by refluxing. DNA normally binds tightly tothe untreated rehydrated Celite surface and is retained during elution.The presence of fluorine causes less tight bonding of DNA to the treatedrehydrated Celite surface, so that the bound DNA would elute from thefluorinated rehydrated Celite surface during the elution step. Ingeneral, as the percentage of fluorine on the rehydrated Celiteincreases, the elution of DNA from the treated surface also increases.Fluorinated rehydrated Celite surfaces prepared by reacting about 0.05equivalent to an excess of fluorine to rehydrated Celite were found toprovide good recovery of DNA from biological samples. It is preferredthat the fluorinated rehydrated Celite surfaces be prepared by reactingabout 0.3 to about 0.9 equivalent of fluorine to rehydrated Celite. ForDNA recovery, several of these fluorinated rehydrated Celite surfacesoutperform super fine super floss Celite.

The fluorinated surfaces of the present invention can be used for thepurification of DNA from other cellular components or potentialcontaminants. The DNA can be obtained from any source, including but notlimited to crude cell extracts, biological fluids, phage supernatants,agarose gels and radiolabelling reactions. The DNA can bedouble-stranded, single-stranded, circular or linear, and can bevariable in size. Conventional techniques for obtaining DNA from anysource, well known in the art, are utilized to prepare the DNA forpurification. Typical procedures for obtaining DNA end with a suspensionof the DNA in solution. For isolation of DNA from biological samples,see, e.g., Harding, J. D. et al., Nucleic Acids Research 17:6947 (1989)and Marko, M. A. et al., Analytical Biochemistry 121:382 (1982).Procedures for isolation of plasmid DNA can be found in Lutze, L. H. etal., Nucleic Acids Research 20: 6150 (1990). Extraction ofdouble-stranded DNA from biological samples can be found in Yamada, O.et al., Journal of Virological Methods 27:203 (1990). Most DNA solutionscomprise the DNA in a suitable buffer such as TE (Tris-EDTA), TEA (40 mmTris-acetate, 1 mm EDTA) buffer, or a lysate. See also Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, New York (1989).

Once the DNA is obtained in a suitable solution or suspension, thefluorinated surface of the present invention is added to the solution orsuspension. Alternatively, the DNA solution or suspension could be addedto the fluorinated surface of the present invention. After the DNAsolution or suspension is contacted with the fluorinated surface of thepresent invention, a binding buffer typically is added to assist in thebinding of the DNA to the fluorinated surface. Suitable binding buffersinclude well-known chaotropes such as NaClO₄ and NaI, and other agentssuch as guanidine HCl or isopropanol. After the DNA is bound to thefluorinated surface, the pure DNA is eluted from the fluorinatedsurface. Suitable eluting agents include water or 10 mM Tris, pH 7.0.Generally, the fluorinated surface with bound DNA is separated, e.g., bycentrifugation or filtration, and washed prior to eluting the DNA.Suitable washing agents include 80/20 ethanol/50 mM Tris, pH 7.0 andother low molecular weight alcohols.

The DNA obtained by purification with the fluorinated surfaces of-thepresent invention may be used without further manipulation forrestriction enzyme digestion, cloning, sequencing, diagnostics and thelike. The high quality of DNA prepared with the present invention andthe speed with which DNA is purified with minimal downstream processingmean that these fluorinated surfaces can be useful in the automation ofDNA sample preparation.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

EXAMPLE 1

Synthesis of Fluorinated Surfaces

A. Fluorinated Al(OH)₃

Fluorinated Al(OH)₃ surfaces comprising Al(OH)₃ reacted with 0.05 to 0.3equivalent of fluoride were prepared using the following ratios oftetrabutylammonium fluoride (TBAF; Aldrich Chemical Co. ) and Al(OH)₃(Aldrich):

    ______________________________________                                        TBAF                    Al (OH).sub.3                                         Rxn    eq.       ml     mMol      g   mMol                                    ______________________________________                                        1      0.3       2.20   2.20      .5  6.67                                    2      0.1       .700   .700      .5  6.41                                    3      0.05      .035   .035      .5  6.41                                    ______________________________________                                    

Reaction 1 was performed by adding the TBAF to the solid Al(OH)₃. Ten mlTHF was then added, and the mixture again was heated with stirring toreflux. Twenty ml THF was added, and the mixture was refluxed withstirring overnight. The reaction mixture was then cooled to roomtemperature and filtered. The fluorinated Al(OH)₃ was washed three timeswith 20 ml water to remove F⁻, and three times with 15 ml acetone. Thewashed material was air-dried for 30 minutes and heat-dried at 100° C.for 30 minutes. The dried, fluorinated Al(OH)₃ was stored in adesiccator.

Reactions 2 and 3 were performed by adding together the TBAF, Al(OH)₃and 10-15 ml THF. A reflux condensor was attached and the reactionmixture was refluxed overnight with stirring. The reaction mixture wasthen cooled to room temperature and filtered. The fluorinated Al(OH)₃was washed three times with 10 ml acetone, three times with 15 ml waterand three times with 15 ml acetone. The washed material was air-driedfor 20 minutes, oven-dried at 100° C. for 1 hour, and stored in adesiccator.

B. Fluorinated Celite

Fluorinated Celite surfaces comprising rehydrated Celite reacted with0.3 equivalent to an excess of fluoride to SiO₂ were prepared by usingthe following ratios of TBAF and Celite (SiO₂):

    ______________________________________                                        TBAF                   Celite*                                                Rxn    eq.       ml     mMol     g   mMol                                     ______________________________________                                        1      0.3       2.8    2.8      .5  8.33                                     2      0.6       5.6    5.6      .5  8.33                                     3      0.9       9.0    9.0      .5  8.33                                     4      exs       12.0   12.0     .5  8.33                                     ______________________________________                                         *Celite 545 was oxidized prior to use by acid washing with HNO.sub.3 or       H.sub.2 SO.sub.4.                                                        

Reaction 1 was performed as described for reaction 1 of the fluorinatedAl(OH)₃ surfaces. Reactions 2-4 were performed as described forreactions 2 and 3 of the fluorinated Al(OH)₃ surfaces.

EXAMPLE 2

Analysis of DNA Recovery Using Super Fine Super Floss Celite as Standard

The following materials were utilized for the analysis of DNA recoverywith super fine super floss Celite as a standard for the analysis of theDNA recovery capabilities of the silicon-containing materials:

Super Fine Super Floss Celite (Manville; 1:5 w/w in H₂ O) [SFSF]

μDNA (BRL Cat. No. 56125A)

50 mM Tris, pH 7.0 (diluted from 1M stock)

Binding buffers (H₂ O or NaClO₄ diluted from 6M stock)

80% ethanol in 50 mM Tris, pH 7.0

MilliQ H₂ O

Ethidium bromide (10 mg/ml)

1% agarose

1X TAE (diluted from 50X stock)

Type II loading dye (25% Ficoll 400, 25% bromophenol blue, 25% xylenecyanol)

Types 57 and 55 Polaroid film

Fifty μl of λ DNA solution (0.5 μl λ DNA in 50 μl 50 mM Tris, pH 7.0,for 31 μg DNA/reaction) were added to eight tubes. Twenty μl of SFSF wasadded to the DNA (˜30 μg). Four hundred μl of binding buffer was addedto the DNA as follows: H₂ O to tube 1; 1.0, 1.5, 2, 2.5, 3, 3.5 and 4MNaClO₄ to tubes 2-8, respectively. The mixture was incubated withrocking for 10 minutes at room temperature. The tubes were centrifugedand the supernatant was discarded. Resulting pellets were washed twicewith 80/20 ethanol/50 mM Tris HCl, pH 7.0. The DNA was eluted from thepellet in 20 μl water for 10 minutes at 37° C. The tubes werecentrifuged and the supernatants of each saved in a separate tube. Thepellets were eluted again, as before, the tubes centrifuged and thesupernatants combined. Two μl of Type II loading dye was added to eachtube of the supernatants and the mixture loaded into a 1% agarose, 1XTAE gel. The gel was run for about 25 minutes at 100-130 volts in 1X TAEbuffer. The gels were stained with ethidium bromide in H₂ O (˜1:1000)for ˜20-30 minutes. Photographs over UV light were taken with Type 57film and negatives were taken (when possible) with Type 55 film.

The gels showed that a small amount of DNA eluted from the SFSF withwater used as the binding buffer. A small amount of DNA was also elutedwith 1, 1.5 and 2.0M NaClO₄ used as the binding buffer. A dramatic risein the amount of eluted DNA was seen with 2.5, 3.0, 3.5 and 4.0M NaClO₄used as the binding buffer. When SFSF was compared with Prep-A-Gene™, itwas seen that no DNA was eluted from the Celite from Prep-A-Gene™ until3.0M NaClO₄ was used as the binding buffer, whereas SFSF bound some DNAin its native state and bound it more strongly at 2.5M NaClO₄. Thus,SFSF performed better than Prep-A-Gene™. In the Examples which follow,SFSF was used as the standard, using 3M NaClO₄ as the binding buffer.

EXAMPLE 3

Analysis of DNA Recovery Using Fluorinated Al(OH)₃

The recovery of DNA using the fluorinated Al(OH)₃ prepared in Example 1was analyzed by following the procedure set forth in Example 2, exceptthat seven tubes contained the fluorinated Al(OH)₃ 20 μl suspension (˜30μg) and 1, 1.5, 2, 2.5, 3, 3.5 and 4M NaClO₄ (400 μl each) was used asthe binding buffer. The eighth tube (control) contained SFSF 20 μlsuspension (˜30 μg) and used 3.0M NaClO₄ as the binding buffer. Thefollowing results were obtained. A fluorinated Al(OH)₃ reacted with 0.3equivalent of fluorine to Al(OH)₃ (reaction 1) showed good recovery(i.e., binding and eluting) of DNA down to 1.5M NaClO₄ (i.e., 1.5MNaClO₄ used as the binding buffer), out-performing SFSF. A fluorinatedAl(OH)₃ reacted with 0.3 equivalent of fluorine gave excellent recoveryof DNA, out-performing Prep-A-Gene™ (see Example 5).

EXAMPLE 4

Analysis of DNA Recovery Using Fluorinated Celite

The recovery of DNA using fluorinated Celite prepared in Example 1 wasanalyzed as described in Example 3. The following results were obtained.A fluorinated Celite prepared by reacting 0.3 equivalent of fluorine torehydrated Celite, prepared by reaction 1 but without refluxing, did notrecover any DNA. A fluorinated Celite reacted with 0.3 equivalent offluorine prepared by reaction 1, eluted some DNA down to 2M NaClO₄. Afluorinated Celite prepared by reacting 0.6 or 0.9 equivalent offluorine, prepared by reaction 2 or 3, eluted DNA down to 1M NaClO₄ withgood amounts eluted down to 1.5M NaClO₄. A fluorinated Celite preparedby reacting excess fluorine prepared by reaction 4, gave DNA recoverydown to 1.5M NaClO₄.

EXAMPLE 5

Analysis of Quantitative DNA Recovery

A 1:10 dilution of λ DNA (500 μg DNA in 658 μl TE buffer (10 mMTris-HCl, 1 mM EDTA, pH 8.0)) was prepared. DNA samples were prepared,each containing 10 μl of the diluted λ DNA and 230 μl TE buffer. Astandard DNA sample was prepared containing 40 μl TE buffer and 10 μl ofthe diluted λ DNA. Thirty μl of Al(OH)₃, fluorinated Al(OH)₃ (reaction 3of Example 1) and Prep-A-Gene™ Celite were added to the DNA samples,followed by 750 μl of Prep-A-Gene™ binding buffer. The samples wereshaken for 10 minutes at room temperature. The samples were centrifugedand decanted. The binding step, including centrifugation and decanting,was repeated. Five hundred μl Prep-A-Gene™ wash buffer was added, andthe samples were shaken for five minutes at room temperature. Thesamples were centrifuged, decanted and dried at 60° C. for 10 minutes.Twenty-five μl of Prep-A-Gene™ elution buffer was added, the samplemixed and then heated at 60° C. for 10 minutes. The samples werecentrifuged and the supernatants combined. Gel electrophoresis wasperformed as described in Example 2, with 3 μl of Type II loading dyeadded to 7 μl eluted DNA. Gel electrophoresis showed that no DNA waseluted from the Al(OH)₃ surface, whereas DNA was eluted from thefluorinated Al(OH)₃ and Prep-A-Gene™ Celite.

The samples were also analyzed by a tri-carb 300 scintillation counter.Three samples from each of the different surfaces were counted todetermine the location of the DNA. These samples were: (1) the originalbinding buffer following the first binding step; (2) the elution bufferafter the second elution step, and (3) the binding matrix (surface). Theanalysis was conducted as follows: (1) two volumes of 6 ml scintillationfluid were added to the binding buffer; (2) 6 ml scintillation fluid wasadded to 40 μl of the elution buffer, and (3) 6 ml scintillation fluidwas added to the binding matrix. This analysis showed that Al(OH)₃removed more DNA (94.9%) from the original solution than the other twosurfaces. However, 99.3% of what was bound remained bound to the surfacefollowing elution. The fluorinated Al(OH)₃ bound 24.2% of the DNA, morethan the Prep-A-Gene™ Celite. 74.6% of the bound DNA eluted from thefluorinated Al(OH)₃. This amount of DNA was greater percentage-wise thanwith the Prep-A-Gene™ Celite.

Fluorinated Celite was also analyzed with the gel electrophoresistechnique. DNA was recovered from the fluorinated Celite prepared byreaction 1 of Example 2.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

What is claimed is:
 1. A method for purifying DNA comprising the stepsof:(a) contacting a suspension containing DNA with a fluorinated surfaceprepared by reacting rehydrated Celite with from about 0.05 equivalentto an excess of fluoride ion under conditions suitable to bind DNA tosaid surface; (b) washing said fluorinated surface having bound DNA; and(c) eluting the DNA from said fluorinated surface.
 2. A method forpurifying DNA comprising the steps of:(a) contacting a suspensioncontaining DNA with a fluorinated surface prepared by reactingrehydrated Celite with from about 0.3 to about 0.9 equivalent offluoride ion under conditions suitable to bind DNA to said surface; (b)washing said fluorinated surface having bound DNA; and (c) eluting theDNA from said fluorinated surface.